Fig. 2

RANKL activates St3gal1 transcription in a c-FOS-dependent way. a Genome browser snapshot displaying a 138 kb region surrounding the ST3Gal1 gene locus, highlighting its core enhancer. ChIP-seq data reveal a prominent FOS binding peak, indicating the region bound by the FOS transcription factor, with additional H3K27ac ChIP-seq tracks comparing BMMs and OCs to show enhanced histone acetylation in OCs, alongside ATAC-seq data demonstrating increased chromatin accessibility at the c-FOS binding site in response to RANKL stimulation. b Chromatin immunoprecipitation (ChIP) followed by PCR confirming the binding of FOS to the ST3Gal1 core enhancer across conditions with and without RANKL treatment, Input DNA was included as a control; RNA polymerase II(Pol II) served as a positive control; Immunoglobulin G (IgG) and no antibody served as negative controls. c Schematic representation of the wild-type (wt) and mutant (mt) ST3Gal1 core enhancer sequences. The FOS binding site (TGACTCA) in the wild-type enhancer is mutated (ATATCGT) in the mutant construct. Both constructs were cloned upstream of a luciferase reporter. d Luciferase reporter assay showing normalized luciferase activity in BMMs and OCs transfected with the wild-type or mutant ST3Gal1 enhancer and co-transfected with either a FOS expression vector or an empty vector (n = 5). Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA, with *P < 0.05, **P < 0.01, ***P < 0.001, N.S. not significant